The intestinal commensal microbiota is required for development of lymphoid tissues and mucosal immunity in vertebrates. However, little is known of the mechanism by which these processes occur. Previous work in rabbits has demonstrated that the intestinal commensal microbiota is required for expansion of B cells and somatic diversification of Ig genes during development of the preimmune antibody repertoire, both of which occur in gut-associated lymphoid tissues (GALT). These processes occur within a few days after birth in an antigen- and T cell-independent manner, and surprisingly, they require activation of complement. We hypothesize a model for GALT development in which B cells enter GALT from the bone marrow soon after birth and migrate to the follicle-associated epithelium (FAE) and interact with C'-coated bacteria from the lumen. These complexes are then delivered to FDCs which provide stimulatory signals for B cell activation and upregulation of AID expression; within a few weeks after birth, essentially all Ig genes are somatically diversified, thereby providing the rabbits with a diverse antibody repertoire. The goal of this grant is to test this model of GALT development. In Aim 1 we will use soluble chemokine receptors to inhibit chemokine-driven migration and determine how the migration of B cells is altered; in Aim 2, we will use sterile GALT explants and TLR and NLR agonists and antagonists to determine the contribution of TLR and NLR ligands and IgM-binding bacterial superantigen-like molecules to B cell proliferation and Ig gene diversification; we will also use sterile explants of GALT as well as an in vitro FDC:B cell culture system to determine how complement activation contributes to GALT development. In Aim 3, we will search in gnotobiotic CD4-/- mice for GALT-like B cells that proliferate in an antigen and T-cell independent manner and undergo somatic diversification of the Ig genes. These experiments are important because they will begin to determine the mechanism by which commensal bacteria and complement contribute to development of mucosal lymphoid tissues and ultimately to human health and disease such as allergy and inflammatory bowel disease. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to determine the mechanism by which intestinal commensal bacteria promote development of gut-associated lymphoid tissues. These studies will provide insight into the origin of bacteria-induced gastrointestinal diseases such as inflammatory bowel disease.